The overall goal of this research is to elucidate the signaling pathways responsible for protective adaptation. Brief periods of intermittent exposure to stress (known as preconditioning (PC)) have been shown to provide protection against injury during a subsequent more severe stress. We recently have investigated several signaling pathways that might be involved in the protection afforded by PC. We tested the hypothesis that a lipoxygenase metabolite might be involved in preconditioning, since lipoxygenase metabolites have been shown to modulate ion channel and transport activity. In support of this hypothesis we find that addition of the lipoxygenase inhibitors NDGA and ETYA during preconditioning, block the protective effects of preconditioning. We further find that 12-HETE, the stable end-product of lipoxygenase metabolism, is made during the preconditioning protocol. Furthermore, we have recently observed that baicalein, an antioxidant which inhibits the 12-lipoxygenase also blocks preconditioning. We also find that alteration in redox state appears to be involved in preconditioning. Preconditioning results in a slight, but significant decrease in the GSH/GSSG, and addition of the antioxidant and glutathione precursor, N-acetyl-cysteine during the preconditioning protocol is found to block the protective effects of preconditioning. Furthermore, diamide and BSO given prior to sustained ischemia mimic preconditioning. We also find that the endogenous PKC activator, DOG, can mimic preconditioning, and that the PKC inhibitor, chelerythrine blocks the protection afford by preconditioning when it is added during the preconditioning protocol. We have recently described the first direct measurement of sarcoplasmic reticulum (SR) ionized calcium concentration. We used an NMR sensitive, high Kd indicator which loads into SR in perfused rabbit hearts. We find that basal SR ionized calcium is 1 mM. We also observed that SR calcium is slightly decreased during preconditioning. Furthermore, in preliminary studies we find that transient addition of agents which reduce SR calcium (CPA, thapsigargin) mimic preconditioning.